Over the past decade a great deal of research has been focused on the development of technology related to micro-total-analytical systems. This technology is based on the concept of a series of microfluidic channels also known as microchannels for the movement, separation, reaction, and/or detection of various materials, such as chemicals or biological compounds like amino acids, proteins, and DNA.
One disadvantage with prior microfluidic devices is that there is frequently a mismatch between the extremely small quantities of sample material used for analysis and the often much larger quantities needed for loading the material into the microfluidic device and transporting it to the point of analysis. For example, a typical analysis sample may be around one nanoliter or less of a liquid containing one or more materials that is injected into a separation channel and then separated electrokinetically as it moves down the channel to a detection region. However, the channels used to transport the sample materials to the injection point are typically also filled with the sample, thus increasing the required amount of the sample by a factor of 100 or more. In addition, the sample is typically loaded onto the microfluidic device into a reservoir from a pipette so that in all, approximately 99.9% of the sample is discarded as waste.
One way of addressing the problem of requiring a large amount of sample material for analysis due to the inefficiencies of conventional devices which result in wasted sample is to use one of any number of focusing techniques to concentrate the materials. In the context of this disclosure, focusing refers to methods for manipulating the velocity of a material and thereby causing it to move towards a position at which the velocity is zero. At the zero velocity position the material will accumulate and increase in concentration, i.e., it will become focused. In addition, the location of the zero velocity position is often dependent upon some characteristic of the material being focused, so that different materials are focused at different positions, and can thereby be separated from a mixture of materials.
Prior focusing methods include isoelectric focusing; electromobility focusing; counteracting chromatographic electrophoresis; temperature gradient focusing, disclosed in U.S. patent application Publication No. 2003/0019752, herein incorporated by reference; and most recently, mixing reactions by temperature gradient focusing, disclosed in co-pending U.S. patent application Ser. No. 10/039,904 herein incorporated by reference; and micellar affinity gradient focusing, disclosed in U.S. patent application Publication No. 2004/0206626, herein incorporated by reference. With the exception of the recently described micellar affinity gradient focusing method, the focusing methods all separate different materials based upon their electrophoretic properties, e.g., mobility in the case of electromobility focusing and temperature gradient focusing, and the isoelectric point in the case of isoelectric focusing.
Electric field gradient focusing is one focusing technique which can be used to concentrate a material at a given position within a microfluidic device before the analysis or other step. Further, the electric field gradient can be used to concentrate all of a selected material at the beginning of the separation channel so that very little of the material would be wasted.
Electric field gradient focusing is accomplished by the application of an electric field gradient within a microchannel. In response to the electric field gradient, there is a corresponding gradient in the electrophoretic velocity of any ionic material within the microchannel. The total velocity of the material is the sum of its electrophoretic velocity and the bulk fluid velocity. If these two components of the velocity are in opposite directions, they can be balanced so that the material will have zero total velocity.
When there is a gradient in the electrophoretic velocity, the balance between the bulk fluid velocity and the electrokinetic velocity can occur at a single position along the microchannel and therefore can result in focusing of the material at that position. Typically, the electric field gradient used in focusing is generated by the external manipulation of the electric field in the middle of the microchannel through the use of conducting wires, salt bridges, porous membranes, or other structures that will pass electric current but will restrict the flow of bulk fluid and the materials that are to be focused.
Several recent developments with regard to focusing methods in microfluidics, and in particular, the use of electric field gradients, have been made. A description of related methods of focusing can be found in C. F. Ivory, W. S. Koegler, R. L. Greenlee, and V. Surdigio, Abstracts of Papers of the American Chemical Society 207, 177-BTEC (1994); C. F. Ivory, Separation Science and Technology 35, 1777 (2000); Z. Huang and C. F. Ivory, Analytical Chemistry 71, 1628 (1999); W. S. Koegler and C. F. Ivory, Journal of Chromatography a 726, 229 (1996); and P. H. Ofarrell, Science 227, 1586 (1985), all of which are hereby incorporated by reference.
To illustrate the basic principles disclosed in these publications, reference is made to FIG. 1(a) which depicts a length of fluid-filled microchannel of constant cross-sectional area with an electrode, denoted 4, in the middle, and two further electrodes at each end, denoted 3 and 5, so that the voltages V1, V3 at the ends and the voltage V2 at the middle of the channel can be controlled. A single species of negatively charged ionic material is present in the fluid that is within the microchannel. The electrical connection, represented as electrode 4, can be accomplished with a simple metal wire as depicted in FIG. 1(a), or through a more complicated structure consisting of additional fluid channels and porous membrane structures or salt bridges.
The electric field in the section 1, i.e., the channel between electrodes 3 and 4 is E1=(V2−V1)/(l/2) and the electric field in section 2, i.e., between electrodes 4 and 5, is E2=(V3−V2) /(l/2), where V1, V2, and V3 are the voltages applied to the three electrodes 3, 4, and 5, and l is the length of the microchannel. If E1 differs from E2 as shown in FIG. 1(b), the electrophoretic velocity of the ionic material in the channel, uEP, will be different in section 1 than in section 2. If an overall bulk fluid velocity, uB<0, is applied, e.g., either electro-osmotic or pressure-driven, the bulk fluid velocity must be the same, due to continuity, in all parts of the microchannel. The total velocity of the ionic material, uT=uB+uEP, will then be the sum of the electrophoretic and bulk velocities, which can differ in section 1 from section 2.
The use of the microchannel device of FIG. 1(a) for focusing of the ionic material is illustrated in FIG. 2 where uT,1>0>uT,2, so that the ionic material moves into the middle from both directions and is thus focused in the middle of the channel near electrode 4.
One major drawback to electric field gradient focusing is that the device tends to be difficult to construct and that it requires the control of voltage on an additional electrode, e.g. 4 of FIG. 1(a), that is used to apply the electric field gradient. In addition, if electrodes are used to generate electric field gradients, unwanted chemical products will be generated electrochemically at the fluid-electrode interface. If the electric field gradient is produced through the use of a salt bridge or membrane, the electrochemical products can be avoided, however only materials or chemical species that cannot pass through the membrane or salt bridge can be focused.
Two additional methods for concentrating a sample include sample stacking and field amplified sample injection in which a sample is concentrated as the sample crosses a boundary between low and high conductivity fluids. These methods can achieve preconcentration factors of 100 to 1000-fold although these methods require multiple fluids. Sweeping is yet another concentration method which is capable of a very high degree of sample concentration (e.g., up to 5000-fold), but is useful only for small molecular weight hydrophobic materials with a high affinity for a mobile micellar phase.
An additional technique for concentrating an ionic material includes isoelectric focusing. Isoelectric focusing is commonly used for the concentration and separation of proteins and involves the focusing of materials at their respective isoelectric points along a pH gradient.
Two examples of recent isoelectric focusing techniques are provided by U.S. Pat. No. 3,664,939 to Luner et al. and U.S. Pat. No. 5,759,370 to Pawliszyn. Both references relate to isoelectric focusing with pH gradients that are created by the application of a temperature gradient. Isoelectric focusing uses a pH gradient to focus materials and in particular proteins, at positions along the pH gradient where the local pH is equal to the isoelectric points of the materials. The isoelectric point is the pH at which the material has zero electrophoretic mobility, i.e., approximately zero charge. pH gradients for isoelectric focusing are typically generated using ampholyte mixtures or immobilized ampholytes in gels. The two above referenced patents are included here as examples of prior art uses of temperature gradients for focusing. It is, however, very unusual for isoelectric focusing to be done with a pH gradient generated using a temperature gradient.
One disadvantage of isoelectric focusing is that it is limited in application because it can only be used with materials having an accessible isoelectric point. Additionally, the concentration to which a protein can be focused with isoelectric focusing is severely limited due to the low solubility of most proteins at their isoelectric points.
In addition, further methods have been developed specifically to separate chiral species. Of the conventional methods of chiral separation, chiral capillary electrophoresis typically gives the best resolution, and is consequently widely used for analytical scale separations of chiral compounds and assays of enantiomeric purity. The major limitation of chiral capillary electrophoresis and of capillary electrophoresis in general is its poor detection limits, which are typically too high for the analysis of chiral compounds, e.g., drugs and drug metabolites in body fluids such as serum or urine. This major limitation is discussed throughout the scientific literature, including Scriba, G. K. E. Electrophoresis 2003, 24, 2409-2421; Bonato, P. S. Electrophoresis 2003, 24, 4078-4094; Hempel, G. Electrophoresis 2000, 21, 691-698; Chen, S. H.; Chen, Y. H. Electrophoresis 1999, 20, 3259-3268; and Altria, K. D.; Elder, D. Journal of Chromatography A 2004, 1023, 1-14.
Capillary electrophoresis is also commonly used to assess the enantiomeric purity of drugs. In order to detect and quantify a trace contaminant of one enantiomer against a large excess of the other, it is often necessary to reverse the order of peak migration so that the tail of the major enantiomer peak does not obscure the small impurity peak.
Isoelectric focusing and isoelectric trapping have also been used in conjunction with chirally selective additives for enantiomeric separations. However, these techniques are limited in their applicability because the chiral materials or molecules to be focused must be chemically derivatized so that 1) they have an isoelectric point, and 2) the isoelectric point is dependant on the degree of interaction with a chiral selector. The vast majority of pharmaceutical molecules do not have isoelectric points and so cannot be focused or separated with isoelectric focusing. Furthermore, most pharmaceutical molecules would not be amenable to the kind of chemical derivatization required for isoelectric focusing, see, e.g., Ogle, D.; Ho, A.; Gibson, T.; Rylatt, D.; Shave, E.; Lim, P.; Vigh, G. Journal of Chromatography A 2002, 979, 155-161; Rizzi, A. M.; Kremser, L. Electrophoresis 1999, 20, 3410-3416; Righetti, P. G.; Ettori, C.; Chafey, P.; and Wahrmann, J. P. Electrophoresis 1990, 11, 1-4 for a further discussion regarding the separation of chiral molecules using isoelectric focusing.